1. Field of Invention
This invention relates to methods and apparatuses for segregating, i.e. separating, purifying and/or concentrating ions in a mixture of different molecular species. More particularly, this invention relates to a combination of electrophoresis and a flowing carrier fluid.
2. Brief Description of the Prior Art
The principles of electrophoresis are known whereby separation of a mixture of ions can be effected by an applied voltage field since different species will migrate toward an oppositely charged electrode at different rates proportional to their charge to mass ratio. It is also known that particular hydrated supports or matrices can have an influence on electrophoresis rates and can be used in conjunction with electrophoresis to effect the separation of ions according to physical properties other than their charge to mass ratio. In addition, a specialized technique is known, in which an electric potential forces complex polyionic molecules to their isoelectric point in a pH gradient.
Electrophoresis is typically used as an analytical tool in biochemical research. When used in this manner, electrophoresis separates components of a mixture and allows certain measurements, e.g. quantification, of the separated components. Usually analytical electrophoresis methods do not include the recovery of the separated components, i.e. the species are both separated and measured in the separation medium and the separated components are recovered only as a mixture or not at all. Several electrophoretic techniques have been developed for preparative use, i.e. to recover the separated components. The simplest of the preparative electrophoresis systems operates on the same principles as the analytical methods, wherein a zone containing a mixture of ion species is applied at the origin and the ions are electrophoresed into or through a supporting matrix whose major importance is to prevent turbulent mixing, i.e. as an anticonvection medium. These techniques include a method for recovering the different ions which are separated by virtue of differing rates of electrophoresis through the supporting matrix. U.S. Pat. No. 3,847,785 is illustrative of such techniques.
These techniques are extremely limited in that they have a very low capacity. Further, because electrophoresis cannot be done at high concentrations of ionic components and because electric heating limits the size of the apparatus, such techniques cannot advantageously be operated on a large scale basis. Moreover, the sample must be in a concentrated form, while recovered isolated components are generally very dilute. Finally, such techniques have the added disadvantage of very limited resolution, i.e. poor capacity to separate two or more very similar components.
Methods of continuous electrophoresis are known in which electrically charged species are continuously fed into an apparatus and are segregated by application of a direct current perpendicular to the flow of a carrier solution. Each charged component moves in a particular direction defined by its rate of electrophoresis in one direction and the rate at which it is carried by the bulk solution flow in the perpendicular direction. Components can thus be collected continuously from the position of zones on the downstream end of the apparatus (see e.g. Ravoo, Gellings and Vermculen, Anal. Chim. Acta 38 (1967) 219-232). By virtue of allowing continuous operation, these techniques have increased the capacity of electrophoretic techniques. However, despite this increase, the capacity is still low, while the other disadvantages of electrophoresis, e.g. poor resolution, remain. Additionally, these continuous electrophoresis processes cannot be adapted to very large scale operation because of inadequate heat dissipation.
Also known is a mode of electrophoresis referred to as isotachophoresis or displacement electrophoresis which was developed following the principles described by Ornstein in U.S. Pat. No. 3,384,564. According to these principles, under particular conditions, an ion of intermediate electrophoretic mobility can be "sandwiched" between an ion with higher electrophoretic mobility and one of lower electrophoretic mobility. Isotachophoresis has the advantage of providing a force which counteracts diffusion and also can concentrate the separated components. However, several problems have been encountered in attempts to apply the method of isotachophoresis. In the absence of spacer ions, the different separated zones of a mixture border on each other and are thus difficult to recover without contamination from adjacent components. Spacer ions must possess very particular properties and thus are not always available. To obtain adequate separation the components must be electrophoresed a considerable distance which demands a high voltage. Also isotachophoresis has a limited capacity.
A method has been described (Preetz and Pfeifer, Anal. Chim. Acta 38 (1967) 255-260) in which bulk fluid flow counteracts isotachophoresis. The purpose of this counter flow is that isotachophoresis can continue for a long time in a small chamber or column. This decreases the power requirements as disclosed in U.S. Pat. No. 3,705,845. Furthermore, bulk fluid flow perpendicular to direction of isotachophoresis has been used to give a continuous method analogous to that described for electrophoresis above.